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. 2016 Feb 29;7(6):601-5.
doi: 10.1021/acsmedchemlett.6b00042. eCollection 2016 Jun 9.

Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7

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Free PMC article

Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7

Chunyan Ren et al. ACS Med Chem Lett. .
Free PMC article

Abstract

The chromobox 7 (CBX7) protein of the polycomb repressive complex 1 (PRC1) functions to repress transcription of tumor suppressor p16 (INK4a) through long noncoding RNA, ANRIL (antisense noncoding RNA in the INK4 locus) directed chromodomain (ChD) binding to trimethylated lysine 27 of histone H3 (H3K27me3), resulting in chromatin compaction at the INK4a/ARF locus. In this study, we report structure-guided discovery of two distinct classes of small-molecule antagonists for the CBX7ChD. Our Class A compounds, a series including analogues of the previously reported MS452, inhibit CBX7ChD/methyl-lysine binding by occupying the H3K27me3 peptide binding site, whereas our Class B compound, the newly discovered MS351, appears to inhibit H3K27me3 binding when CBX7ChD is bound to RNA. Our crystal structure of the CBX7ChD/MS351 complex reveals the molecular details of ligand recognition by the aromatic cage residues that typically engage in methyl-lysine binding. We further demonstrate that MS351 effectively induces transcriptional derepression of CBX7 target genes, including p16 (INK4a) in mouse embryonic stem cells and human prostate cancer PC3 cells. Thus, MS351 represents a new class of ChD antagonists that selectively targets the biologically active form of CBX7 of the PRC1 in long noncoding RNA- and H3K27me3-directed gene transcriptional repression.

Keywords: Chromodomain; antagonist; gene transcription; polycomb repressive complex.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structure-guided optimization of MS452 series antagonists for the CBX7ChD. (A) Crystal structures of CBX7ChD bound to MS452 (also known as MS37452) in cis or trans conformation. Right, details of molecular interactions of trans-MS452 bound to CBX7ChD. (B) In vitro SAR study of MS452 and its chemical analogues.
Figure 2
Figure 2
Structure-enabled discovery of MS351 as a novel CBX7ChD antagonist. (A) NMR spectral characterization of MS351 binding to the CBX7ChD, as assessed by 2D 1H–15N HSQC spectra of the 15N-labeled CBX7ChD (200 μM) depicting chemical shift perturbations of protein residues upon addition of MS351 (200 μM). Residues exhibiting major perturbations are annotated. (B) Fluorescence anisotropy assay assessing relative binding affinity of CBX7ChD (120 μM) to RNA, H3K27me3 (aa 21–33), SETDB1-K1170me3 (aa 1165–1174), MS452, or MS351. A FITC-labeled loop C of ANRIL (10 nM) was used as an assay probe as described in detail in our previous study. (C) Dose-dependent effects of MS351 on transcriptional derepression of p16INK4a after the 12 h compound treatment (1–5 μM) in human prostate cancer PC3 cells. HIF1a, a non-CBX7 target gene, was used as a control in this study. (D) Upper panel, immunoblots of p16INK4a of mouse embryoid bodies (EBs) following 8 days of leukemia inhibitor factor (LIF) in the presence of MS351. Lower panel, p16INK4a mRNA level measured by qPCR with TaqMan probe set of RNA obtained from mouse embryonic stem cells treated with or without MS351. (E) Upper panel indicates enrichment of Cbx7 in mouse embryonic stem (ES) cells. Alignments were plotted on UCSC Genome tracks to depict chromosome 2 and enrichment along the axis of chromosome 2 directed from the centromere based on existing Cbx7 ChIP-Seq data. Enrichments are valued as peak calls through alignments of redundant sequence reads using BOWTIE to create WIG files and plotted using GALAXY and cis-genome software algorithms. Lower left panel, qPCR to measure Hoxd3 and Noggin mRNA following treatment of MS351 (0.5 μM) at the time points indicated. Lower right panel, direct ChIP analysis of Cbx7 and histone H3 at the transcript start site of mouse Hoxd3. Gapdh, a non-CBX7 target gene in mESCs, was used as a control for both qPCR and ChIP experiments.
Figure 3
Figure 3
Molecular basis of MS351 recognition by the CBX7ChD. (A) Comparison of crystal structures of the CBX7ChD binding to MS351 (yellow) or H3K27me3 peptide (magenta) in the aromatic cage. The protein is colored in pink and cyan for the MS351 or H3K27me3-bound form, respectively. (B) Structural insights of MS351 interactions with Thr41 and Glu43 of β3 strand that bridges the H3K27me3 binding aromatic cage and noncoding RNA ANRIL binding site involving residues located on the β1′−β2−β3 sheet. Note that Arg17 was identified to be one of the key residues essential for ANRIL binding. (C) Schematic illustration of the role of CBX7 of the PRC1 in noncoding RNA ANRIL and H3K27me3-mediated gene transcription, and the distinct modes of action of MS452 (blue hexagon) and MS351 (purple pentagon) in modulating CBX7 in this functional context.

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